We propose to investigate the post-binding events related to FSH action in Sertoli cells from immature and mature rats. The focus of this project will be on the GTP binding proteins associated with adenylate cyclase activity and the cAMP binding proteins (such as the regulatory subunits of Type I and Type II protein kinase) which regulate the intracellular processes of Sertoli cells. Both of these areas center on cAMP as the mediator of the action of FSH on Sertoli cell function. In each case the nucleotide protein interaction may play pivotal roles in signal transduction (FSH - cAMP - Biological Response). We will be using two photoaffinity probes, [32P]8-N3GTP and [32P]8-N3 cAMP to: a) label and identify GTP binding proteins in membranes of cultures Sertoli cells, b) determine the site of GTPase activity and hormone sensitivity of the GTPase, c) determine the ability of 8-N3GTP to stimulate Sertoli cell adenylate cyclase activity d) examine the modulation of GTP binding sites in relation to FSH stimulation, e) examine the developmental changes in GTP binding proteins in relation to receptors for FSH and adenylate cyclase activity, f) examine the role of GTP binding proteins in the mechanism of hormone induced desensitization of the adenylate cyclase, g) label and identify cAMP binding proteins in cultured Sertoli cells and their subcellular fractions, h) examine the intermediate role of cAMP in FSH induced estradiol and androgen binding protein production and protein phosphorylation. Experimental approaches will rely extensively on past studies with these photoaffinity analogs in erythrocyte membranes and luteal cell homogenates. We will exploit the ability to study several binding sites at once by using a combination of SDS gel electrophoresis, autoradiography and liquid scintillation counting. Previous studies have indicated that the 'cold trap' technique should allow us to quantitate nucleotide binding sites under various experimental conditions.